Topic: Tox / TDM / Endocrine
Podium Presentation in Room 1 on Wednesday at 16:35 (Chair: Mark Marzinke / Ganesh Moorthy)
Authors: Stacy Dee (1), Christopher M. Shuford (1), Julia Hannon (1), Matthew L. Crawford (1), Russell P. Grant (1)
Extraction of immunosuppressants (Tacrolimus, Sirolimus, Everolimus, and Cyclosporine A) from whole blood has been well documented in the literature; however, no consensus exists on the solvent, concentration of ZnSO4, or mixing/lysis times and temperatures. Herein, we aim to empirically optimize each of these conditions and determine their impact on the accuracy and imprecision of measurement.
Two methods studied used 400 µL of lysis solution with labeled internal standards added to 50 µL of sample prior to vortex mixing, centrifugation and analysis of the supernatant by LC-MS/MS. An initial, generic method employed a 70% acetonitrile lysis solution with 8 mM ZnSO4 and 30 sec mix at room temperature. The optimized method used a 70% acetonitrile lysis solution without ZnSO¬4 and a 60 min mix at 37oC. Studies used lysed whole blood calibrators spiked with stocks material purchased from Cerilliant®, lyophilized calibrators and controls from ©Recipe, and proficiency materials from LGC.
Using the generic method, within-run imprecision (N>19) was 18.9 – 34.7% for controls and whole blood with the generic method, due to variation in analyte recovery given the internal standard response was precise (CV ≤ 8.0%). Calibration drift was also observed for both lysed and lyophilized calibrators, with the bracketing curves having -46.2 to -24.4% lower results. Proficiency samples showed significant error relative to target values with both lysed and lyophilized calibrators (biases: -29.8 and +212.7%; Z-scores of -3.5 to +20.7).
Evaluation of mixing time showed that maximum recovery did not occur by 2 hours at room temperature and was unaffected by ZnSO4 (0-100 mM). Mixing at 37 oC in the absence of ZnSO¬4 showed maximum analyte response at 5 – 10 minutes and stable A:IS ratios (48 – 96% higher than room temperature) after 60 minutes for lysed calibrators, whole blood patient samples, external proficiency samples, as well as lyophilized calibrators and controls.
The optimized method showed within-run imprecision of 1.7 – 4.2% and agreement between bracketing calibration curves (biases of -1.0 to 7.6%), yet lyophilized calibrators provided higher sample results than lysed calibrators for 3 analytes (biases of 18.4 to 29.8%) and lower results for Cyclosporine (bias: -11.6%). Analysis of proficiency samples with lysed calibrators indicated accurate measurements (Z-scores of -1.9 to +0.5).
Improvements in precision and accuracy in the optimized method is explained by enabling analyte extraction to reach a maximum/stable recovery. Incomplete extraction likely explains the variability in the generic method and may explain the large inter-laboratory variance observed in proficiency testing programs. Future experiments will focus on elucidating the source of bias between lyophilized and lysed calibrators, despite common traceability.
|Salary||yes||Laboratory Corporation of America|
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||